Hindawi Publishing Corporation Journal of Pregnancy Volume 2011, Article ID 548171, 11 pages doi:10.1155/2011/548171

Review Article The Role of Placental in Human Fetal Growth Restriction

Padma Murthi,1, 2 Gayathri Rajaraman,1, 2, 3 Shaun Patrick Brennecke,1, 2 and Bill Kalionis1, 2

1 Department of Obstetrics and Gynaecology, The University of Melbourne, Melbourne, Victoria 3010, Australia 2 Pregnancy Research Centre, Department of Perinatal Medicine, The Royal Women’s Hospital, Parkville, Victoria 3052, Australia 3 Monash Institute of Medical Research, Clayton, Victoria 3168, Australia

Correspondence should be addressed to Padma Murthi, [email protected]

Received 23 November 2010; Accepted 17 February 2011

Academic Editor: David F. Lewis

Copyright © 2011 Padma Murthi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Fetal growth restriction (FGR) is an adverse pregnancy outcome associated with significant perinatal and paediatric morbidity and mortality, and an increased risk of chronic disease later in adult life. One of the key causes of adverse pregnancy outcome is fetal growth restriction (FGR). While a number of maternal, fetal, and environmental factors are known causes of FGR, the majority of FGR cases remain idiopathic. These idiopathic FGR pregnancies are frequently associated with placental insufficiency, possibly as a result of placental maldevelopment. Understanding the molecular mechanisms of abnormal placental development in idiopathic FGR is, therefore, of increasing importance. Here, we review our understanding of transcriptional control of normal placental development and abnormal placental development associated with human idiopathic FGR. We also assess the potential for understanding transcriptional control as a means for revealing new molecular targets for the detection, diagnosis, and clinical management of idiopathic FGR.

1. Introduction intelligence quotients [9, 10]. While FGR can be attributed to obvious fetal (e.g., chromosomal abnormalities), placental 1.1. Fetal Growth Restriction. The regulation of fetal growth (e.g., obvious infarcts), maternal (e.g., tobacco smoking), is multifactorial and complex. Normal fetal growth is deter- and environmental factors (e.g., viral infections), about mined by the genetically predetermined growth potential 70%ofcasesdonothaveaknowncauseandaretermed and further modulated by maternal, fetal, placental, and idiopathic FGR. Idiopathic FGR is frequently associated with environmental factors [1]. Fetal growth restriction (FGR), placental insufficiency [11]. Cordocentesis studies (sampling also known as intrauterine growth restriction (IUGR), is of umbilical fetal arterial or venous blood) show features afailureofthefetustoreachitsfullgrowthpotentialfor consistent with chronically inadequate transplacental oxygen gestation age. FGR is commonly defined as a birth weight of exchange between the mother and FGR fetus [11]. Clinical less than the 10th percentile for gestation, together with evi- features of idiopathic FGR pregnancies include abnormal dence of fetal health compromise such as oligohydramnios umbilical artery Doppler velocimetry [12], oligohydramnios and asymmetric fetal growth involving an increased head to [13], and asymmetric fetal growth [14]. abdominal circumference ratio. Evidence of such underlying pathology allows clinicians to discriminate between FGR 1.2. Pathology of Placental Dysfunction in FGR. Typically, and healthy small for gestation age (SGA) babies that are the placentae in idiopathic FGR are smaller than their otherwise normal. FGR is associated with an increased gestation age-matched controls [15], and they show obvious risk of perinatal complications such as prematurity [2], morphological defects. Macroscopic placental lesions [12] stillbirth [2–5], neonatal morbidity [5, 6], and mortality are frequently evident, whilst microscopic defects such [5, 6]. Adverse outcomes for FGR neonates include impaired as reduced trophoblast proliferation and abnormal villous neuropsychological development [7, 8] leading to reduced vasculature with shorter, less branched terminal villi [16] 2 Journal of Pregnancy are also observed. Another significant functional defect is morphogenesis (reviewed in [32–34]). These genes contain uteroplacental ischemia due to failure of the placental extrav- a highly conserved 180 homeobox sequence, which illous cytotrophoblast cells to effectively carry out the critical encodes a 60 amino acid homeodomain. Structural analyses processes of invasion, transformation, and remodeling of the have shown that the homeodomain consists of an evolu- spiral arteries in the maternal decidua [17]. tionarily conserved helix-turn-helix motif that binds to the At the cellular level, trophoblast function is modulated DNA. The specificity of this binding allows homeodomain in an autocrine/paracrine manner by growth factors, their to activate or repress the expression of batteries of binding proteins, and extracellular matrix components of downstream target genes [35]. the placenta (reviewed in [18, 19]). This modulation of tro- Most important is that homeobox genes are directly or phoblast cell function involves various extracellular signals, indirectly involved in a variety of developmental disorders, signalling molecules, and consequent activation in diseases, and cancers (reviewed in [36]). Homeobox genes the signalling pathway. Disruption of various important are subdivided into the “clustered” homeobox genes known signalling pathways is observed in placental pathologies that as “HOX” genes, the “nonclustered” divergent or orphan are associated with abnormal trophoblast function [20]. HOX-like genes, as well as several distinct classes of atypical A consequence of altered placental function in idiopathic homeodomain containing genes. The HOX family plays FGR is reduced transfer of oxygen, nutrients, and growth a fundamental role in the embryonic morphogenesis and factors to the fetus, which restricts fetal growth [21]. The were identified in mammals and vertebrates based on their changes observed in FGR placentae are consistent with early to the genes of the Drosophila HOM-C developmental defects [17], but the developmental genes [37, 38]. In mice and humans, the HOX complex is com- involved and their molecular mechanism of action are not prised of 39 genes that are arranged into four separate chro- known. Several longitudinal studies have demonstrated a mosomal clusters designated HOX A, B, C, and D [39, 40]. possible causative role for genetic and familial factors, as yet Homeobox genes are grouped together into various unidentified, in human FGR [22, 23]. subfamilies based on a variety of criteria such as their Current knowledge of the molecular mechanisms of FGR functional and structural characteristics, and these subfam- is limited. Various attempts to understand the molecular ilies of homeobox genes are essential for the control of basis of FGR using microarray and proteomics approaches specific aspects of cellular growth and differentiation [28, have revealed significant differences between FGR and 29, 41]. Evidence for the deregulation of certain homeobox uncomplicated control [24–26] on term placentae and have genes in cancer and other diseases provides support for shed little light on the regulatory mechanisms that underlie the idea that homeobox genes are vital for normal mam- the early events leading to idiopathic FGR. Murine model malian development. Furthermore, characterisation of such systems, particularly those amenable to genetic manipu- homeobox genes may lead to a greater understanding of the lation, are therefore of crucial importance in revealing developmental mechanisms, which are disrupted in a variety potentially important regulatory genes that may play a role of disease states. There is evidence that normal homeobox in the early stages of human FGR. In many animal model expression can be altered during a diseased state, such systems, early developmental stages are controlled at the level as decreased expression of Cdx2 in the intestinal epithelium of transcription factors. of patients with colorectal cancers and decreased Meox2 expression in brain endothelial cells of patients affected 2. Transcriptional Control of by Alzheimer’s disease [36, 42]. Thus, homeobox genes Placental Development could be used as disease markers or potential therapeutic targets of diseases, such as cancer, diabetic wound heal- Growth factors and signalling molecules represent the cue to ing, lymphedema, Alzheimer’s disease, and stroke due to which a cell responds by either maintaining or altering its atherosclerosis [43–45]. state of differentiation [27]. However, it is the transcription Homeobox gene mutations have also been shown to factors, located within the cell nucleus, which determine cause human congenital disorders such as Waardenburg’s how this cue is interpreted and what the cellular response syndrometype1[46, 47] and Aniridia [48]. The homeobox will be. Transcription factors achieve this by regulating gene HuP2 has been found to be mutated in patients with expression of their target genes within the cell. A large Waardenburg’s syndrome [46, 47], and the congenital eye number of different transcription factors play essential roles disorder Aniridia caused by a mutation in the homeobox in cellular development and differentiation of various cell gene designated AN [48]. types, including the trophoblast cell type in the placenta The clustered homeobox genes, known as HOX, play [28, 29]. Transcription factors are categorised into a few large a fundamental role in embryological morphogenesis. HOX families such as the zinc finger, , helix-loop- gene mutations are implicated in various human malforma- helix, helix-turn-helix, and homeobox genes [30, 31]. tions such as hand-foot-genital syndrome, Mowat-Wilson Syndrome, and Duanes Retraction Syndrome (reviewed in 3. Homeobox Genes [36]). There is also an association between mutation in HOX genes and autism spectrum disorders [49]. More recently, Homeobox genes (also known as homeotic genes) were the Aristaless-related homeobox gene, ARX, was found to originally discovered in the fruit fly Drosophila, where be associated with both X-linked mental retardation and they act as transcriptional regulators to control embryonic epilepsy [50, 51]. Journal of Pregnancy 3

Mouse knockouts have also provided genetic proof that 3.2. A Strategy for Understanding Transcriptional Control homeobox genes regulate embryonic organogenesis and in Normal and FGR-Affected Placentae. Our strategy for morphogenesis [52–54]. For example, targeted disruption understanding the molecular mechanisms of placental func- of the Hlx homeobox gene (the homolog of human HLX) tion in normal and FGR-affected human placentae involved in the mouse shows that Hlx plays a fundamental role (i) determining the spatiotemporal expression pattern of in visceral organogenesis [55].Studieshavedemonstrated homeobox genes during placental development that have that Hlx mutant mice resulted in developing gut and liver an “evolutionary history” of regulating cell fate decisions diverticulum defects. In addition, Hlx mutation also showed during embryonic or adult development, (ii) determining a defect in cell proliferation and resulted in embryonic death whether specific homeobox gene expression levels were duetoliverfailure[55]. Furthermore, Hlx is expressed in changed in FGR-affected placentae compared with gestation mesenchymal cell types during organogenesis in the mouse matched controls, (iii) creating in vitro models of placental placenta [56]. Additionally, recent studies from our labora- cultured cells that “mimic” homeogox gene expression tory have confirmed that placental morphology is severely changes observed on FGR by the use of loss- or gain-of affected in Hlx mutant mice (Murthi et al. unpublished data). function phenotypes using RNA interference systems or gene overexpression plasmids, and (iv) defining the biological 3.1. Homeobox Genes in Murine Placental Development. functions of the target genes using in vitro models. These Given the highly important role of homeobox genes in approaches have been proven very successful in identifying embryonic and adult development, it is not surprising transcriptional control of endocrine functions during mouse that homeobox genes also play major roles in controlling placental development (reviewed in [28, 29]). Therefore, extraembryonic development of the placenta. Homeobox identification of the homeobox target genes in specialised genes regulate mouse placental cell functions and targeted cell types of the human placenta can reveal the molecular gene mutations of homeobox genes in the mouse pro- pathways responsible for important placental cell functions. duce FGR-like effects. For example, homeobox gene mouse Thesepathwaysmaybeaffected in FGR. Using this novel mutants, Esx1 and Dlx3, produce FGR-like effects in mice approach, more recent studies in our laboratory have including restricted fetal growth and placental defects [57, described a potential role for transcriptional control of 58]. Esx1 expression is restricted to the placenta and is homeobox gene HLX in the human placental trophoblast not expressed in the embryo. Thus, in the Esx1 mutant cells. In the following section, we will summarise our mouse, altered placental function is the cause of restricted current understanding of homeobox gene HLX regulation in fetal growth. Dlx3 and Esx1 mutant mice show specific human placental development, more specifically to human defects in the labyrinthine trophoblast of the chorioallantoic extravillous trophoblast function, as well as give insights into placenta [57, 58]. In addition, the 3 beta-hydroxysteroid novel mechanisms of trophoblast dysfunction observed in dehydrogenase gene (3β-HSD), which is important for the FGR-affected pregnancies. biosynthesis of all active steroid hormones, is a target of the Dlx3 homeobox gene in the mouse [59]. Therefore, (i) Spatiotemporal Expression Patterns of Homeobox Genes in homeobox genes control important trophoblast functions in the Placenta. Studies in the human placenta have focused the mouse placenta. mainly on identifying homeobox genes expressed in the The homeobox gene Cdx2 is expressed in the embryonic normal placenta [65, 66], and those showing altered expres- trophectoderm and in the spongiotrophoblast component of sion in trophoblastic cancers [67]. The homeobox genes we the placenta at later stages of development and is implicated and others have identified to be of potential importance in the patterning of the extraembryonic membranes of in the human placenta are DLX3 [59, 68, 69],DLX4 the mouse placenta [60]. The finding that Cdx2 homozy- [70–72], MSX2 and GAX [70], ESX1L [58, 73], and gous null mutant mice die between 3.5 and 5.5 days as HLX [74–77]. These genes are potential candidates for a consequence of failed implantation suggests that this regulating epithelial-mesenchymal cell interactions in the homeobox gene may play a role in controlling trophoblast human placenta. These genes are potential candidates for differentiation [61]. regulating epithelial-mesenchymal cell interactions in the The placenta specific–homeobox gene (Psx) also affects human placenta. These genes are also expressed in the mouse placental development. The Psx transcript was first embryo and play major roles in embryonic development [78, detected at embryonic day 8.5 and expression persisted until 79]. Microarray expression profiling of placental trophoblast birth. Psx mRNA is expressed in extraembryonic tissues, and endothelial cells revealed that novel placental homeobox mainly in the placenta, but not in the fetus [62]. Further genes TGIF, MEIS2E, LIM2,andSMAP31-12 are also highly studies have shown that the Psx homeobox gene plays a expressed in trophoblast cells (Murthi et al. unpublished unique role in the function of differentiated trophoblast cells data). in the murine placenta [63]. Few functional studies have been carried out on human Mouse homeobox gene knockouts have also provided placental homeobox genes. One limited study reported that evidence that homeobox genes regulate vascular develop- the inactivation of homeobox gene DLX4 resulted in altered ment and angiogenesis in the mouse placenta (reviewed in rates of trophoblast cell apoptosis [72]. Homeobox gene [29, 41, 64]). Therefore, in animal model systems, homeobox DLX3 regulates the expression of the alpha subunit of hCG genes control trophoblast and endothelial cell functions [59]andof3-βHSD [69], both of which are important for during placental development. placental trophoblast function. 4 Journal of Pregnancy

3.3. Homeobox Genes in Human Placental Endothelial Cells. both HLX and ESX1L expression was observed from 27-week Knowledge of homeobox genes in human endothelial cells gestation, which may correspond to the decline in the growth comes primarily from studies in the cardiovascular system rate of the fetus seen in the third trimester [81, 86]. employing cell culture models such as human umbilical vein Our studies represented the most comprehensive and endothelial cells (HUVEC). Homeobox genes are critical extensive analyses of homeobox genes in placental patholo- regulators of cardiovasculature development [80]. GAX is a gies undertaken. However, homeobox gene DLX4 showed negative regulator of angiogenesis [81]. HOXB3 promotes increased expression [71] in FGR-affected placentae. Our invasive behaviour of endothelial cells in response to angio- observation of altered homeobox gene expression levels, genic stimulation [54], whereas HOXD3 promotes capillary that is, decreased (HLX) or increased (DLX4) expression in morphogenesis [82]. In HUVEC stimulated with VEGF, HEX FGR-placentae compared with gestation matched controls, acts as a negative regulator of angiogenesis [83]. Also in prompted us to identify the downstream target genes which HUVEC, GAX is an inhibitor of endothelial cell activation would be affected by changed homeobox gene levels. in response to growth factors and tube formation [53]. Previous studies from our laboratory have demonstrated (iii) Creating In Vitro Models of Placental Cultured Cells That the expression of homeobox genes HLX, DLX3, DLX4, “Mimic” Homeobox Gene Expression Changes Observed on MSX2,andGAX in placental endothelial cells, and we FGR. Homeobox gene HLX is the most characterised in the showed that novel placental homeobox genes, such asTLX1, human placenta. The HLX gene (also known as HLX1, H2.0- TLX2, TGIF, HEX, PHOX1, MEIS2, HOXB7, and LIM6 were like homeobox or HB24; OMIM 142995) is a member of the also expressed in placental endothelial cells [84]. Our find- homeobox family of genes, with homology to the Drosophila ings have highlighted the potential importance of these genes homeobox gene H2.0. A comparison of HLX orthologs in in the fundamental process of placental angiogenesis. Clearly, human and mouse showed that the genes share similar homeobox genes are important regulators of endothelial cell organization, with four exons and three introns and 85.4% functions in the embryo and adult but their role in placental identity between the human and mouse proteins, suggesting endothelial cells is yet to be determined. a similar function in both species [87]. The HLX homeobox gene was shown to have high expression in haematopoietic (ii) Homeobox Gene Expression Levels Are Changed in FGR progenitor cells, and lower expression levels in activated Placentae Compared with Gestation-Matched Controls. Pre- lymphocytes [88]. vious studies from our laboratory determined the expression Our studies demonstrated that HLX is expressed pri- levels of several homeobox genes in a clinically well-defined marily in the proliferating cytotrophoblast cell types in early idiopathic FGR-affected placentae and gestation-matched placental development and suggested that reduced levels of controls [71, 73, 75]. The cohort of FGR-affected pregnancies HLX are required for cytotrophoblast differentiation and that was employed was carefully defined in clinical terms that dysregulation of HLX may result in aberrant cytotro- and represented the severe end of spectrum of idiopathic phoblast proliferation and differentiation, contributing to FGR. The general inclusion criterion for FGR cases was a placental pathologies [74]. birth weight less than the 10th centile for gestation age, Furthermore, to identify the functional role of reduced using Australian growth charts. FGR cases were classified as HLX levels observed in FGR, we simulated reduced idiopathic if there was evidence of an underlying pathology, expression levels in extravillous trophoblast derived cell judged by the presence of at least two of the following antena- lines SGHPL4 and HTR8-SV neousing short-interference tal ultrasound diagnostic criteria: abnormal umbilical artery RNA (siRNA) specific for HLX. These two transformed Doppler flow velocimetry, oligohydramnios as determined trophoblast-derived cell lines are well-characterized first by amniotic fluid index (AFI) <7, or asymmetric growth of trimester-derived human extravillous cytotrophoblast cell the fetus as measured from the HC (head circumference) to lines and are capable of proliferation, migration, and inva- AC (abdominal circumference) ratio (>1.2). Fetuses showed sion. The results from this study were not cell line specific, reduced growth by the late second and early third trimester. since consistent effects were seen in both the cell lines tested. Reduced villous tree elaboration, diminished surface area Our findings provided evidence of HLX regulation of the placenta, and abnormal end-diastolic blood flow in by cytokines, CSF-1, and growth factors such as HGF, the umbilical artery are characteristic of pregnancies with and established that HLX is an important regulator for severely growth-restricted infants [15, 16]. Homeobox genes signal transduction mediated proliferation and migration of HLX [75]andESX1L [73] showed decreased expression in human extravillous trophoblast cells [76, 77] suggesting that FGR-affected placentae compared with matched controls. HLX may be of pathological significance. The pattern of normal human fetal growth is complex. Increases in the rates of fetal weight gain and length increase (iv) Defining the Biological Functions of the Target Genes are not parallel throughout pregnancy. Evidence suggests Using In Vitro Models. Understanding the precise regula- that the maximal growth rate for length is seen in the second tory mechanisms through which homeobox genes achieve trimester, whereas the maximal rate of weight gain is early molecular control during placental development requires in the third trimester [75, 83]. Guihard-Costa et al. [85]in the identification of target genes within the downstream a longitudinal study of human fetal growth have reported a developmental pathways. linear growth rate until 26 weeks and, thereafter, the growth Genes involved in regulating cellular mechanisms such rate decreased. In our studies, a rapid decline in the levels of as mitotic rate, cell-cell adhesion, and cell migration during Journal of Pregnancy 5 morphogenesis have been identified as target genes for many knockout of RB1 causes embryonic lethality resulting from homeobox genes [89–99]. Thus, homeobox genes act as defects in placental function [106]reviewedin[107]. Wu “master regulators” of development and control transcrip- and coworkers [106] have demonstrated that reduction of tion by binding to regulatory elements in the promoter RB1 gene expression in the mouse model system resulted in regions of target genes [38]. excessive proliferation of trophoblast cells and a severe dis- In the last two decades, the purification, cloning, and ruption of the normal labyrinth architecture in the placenta. characterisation of several homeobox transcription factors, This was accompanied by a decrease in vascularisation and together with transgenic mouse models, have increased our a reduction in placental transport function and ultimately knowledge of the molecular basis of placental development. embryonic death [106]. Whilst loss-of-function studies in the mouse model clearly Our findings demonstrated that RB1 is a direct or demonstrate that homeobox genes such as Cdx2, Cdx4, indirect downstream target of the homeobox gene HLX in Hoxa13,anddlx3 are critical for murine placental devel- cultured human trophoblast cells [108]. Furthermore, RB1 is opment [60, 68, 100], the target genes regulated by these expressed in the proximal region of proliferating trophoblast homeobox genes have not been investigated in either the cells in the trophoblast cell column [109], where HLX is murine or human placenta. also expressed [74]. This provided supporting evidence HLX may act as a regulator of RB1 in trophoblast cells and 3.4. Identification of HLX Target Genes. Previous studies that HLX-mediated RB1 expression in trophoblast cells may showed that inhibition of HLX by antisense oligonucleotide reduce trophoblast proliferation. We also observed that RB1 methods impaired CD34+ bone marrow cell proliferation showed the highest relative increase in expression levels in response to stimulation by cytokines, whilst inducing in FGR-affected placentae compared with control placentae differentiation of these cells. Moreover, HLX inhibition also [108]. These data suggest RB1 is a negative regulator of cell reduced the levels of c-, c-fos, cyclin B,andp34cdc2 proliferation and that increased RB1 expression levels in FGR mRNA expression [88]. These cell cycle regulatory genes may reduce trophoblast proliferation and result in a fewer were predicted to be involved in the function of trophoblast number of trophoblast cells available to migrate and invade cells [101]. By using siRNA-mediated inactivation of HLX into the maternal decidua. This reduction in trophoblast approach, we investigated the mechanisms by which HLX proliferation may also lead to the shallow, inadequate mediates extravillous trophoblast function in normal and remodeling of the maternal spiral arteries associated with FGR-affected placentae. We used siRNA in trophoblast FGR. in vitro models such as SGHPL-4 and HTR-8/SVneo MYC is a proto-oncogene that is overexpressed in a wide and detected changes in gene expression using pathway- range of human cancers. This cell cycle regulator gene is part specific low density PCR arrays for MAP- (mitogen-activated of the postreceptor intracellular signaling pathway for regula- signaling-)kinase signaling pathways. The downstream tar- tion of cell proliferation by growth factors [110]. Depending get genes of HLX were identified as RB1, MYC, EGR1, on the cellular context, MYC proteins induce either cell CDKN1C, ELK1, CCNB1,andJUN. These findings were proliferation or apoptosis and they require cooperation with further validated suggesting the observations were not only other oncoproteins and inhibition of apoptotic pathways to consistent in two independent trophoblast cell lines, SGHPL- transform cells [111]. Previous studies have determined that 4 and HTR-8/SVneo, but was also reflected in FGR-affected MYC is expressed in the actively proliferating extravillous human placental tissue. Most importantly, we identified four trophoblast cells of the human placenta [112–114], where HLX downstream target genes CCNB1, MYC, CDKN1C,and we have shown HLX to be highly expressed [74]. Results JUN, which were previously identified as HLX target genes from our study demonstrated that MYC mRNA expression in haematopoietic progenitor cells [88]astargetsofHLX was significantly increased with HLX inactivation in cultured in cultured trophoblast cells. Thus, HLX homeobox gene trophoblast cells, suggesting that MYC is a direct or indirect targets cell cycle regulatory genes in two independent cell downstream target gene of HLX [108]. types. Targeted disruption of c-myc gene (homolog of MYC) In the following section, we have described further anal- in the mouse model system resulted in severe placental yses of candidate downstream target genes of HLX and their defects including morphological abnormalities [115]. Sev- level of expression and potential contribution to functional eral embryonic developmental defects were also reported abnormalities observed in FGR-affected placentae. including abnormalities in the heart, liver, and neural tube Retinoblastoma-1 (RB1, also known as Rb)isatumor formation. More importantly, embryonic death was also suppressor gene that was first discovered in genetic studies of observed in c-myc knock-out mice due to placental insuf- hereditary retinoblastoma [102]. RB1 also has a role in other ficiency [115]. Our findings showed that MYC expression cancers including osteosarcoma and plays an important role was significantly increased in FGR-affected human placentae, in regulating cell proliferation and differentiation [103]. The consistent with the increase in MYC expression in HLX product of the RB1 gene is a nuclear phosphoprotein that inactivated cultured trophoblast cells [108]. This suggested may act as an inhibitor of cell proliferation [104]. Addition- that MYC, as a downstream target gene of HLX,isa ally, Schubert et al. [105] have demonstrated that RB1 is a molecular target associated with idiopathic human FGR. downstream target of the GCMa/Gcm1 Therefore, HLX-mediated increase in MYC expression may in the mouse placenta. Therefore, RB1 has been shown to be a contribute to increased apoptosis that is frequently associated direct target of transcription factors. In mice, the constitutive with FGR. 6 Journal of Pregnancy

CDKN1C (p57/kip2) is a member of the CIP/KIP family CCNB1 is a regulatory gene expressed predominantly of cyclin-dependent kianse inhibitors and has been shown during the G2/M phase of the cell cycle, functionally involved to inhibit several cyclin-dependent kinase kinase/cyclin in cell mitosis. Studies have shown expression of CCNB1 in complexes and is a regulator of cell proliferation [116]. the villous trophoblast and the extravillous trophoblast cell Mutations of CDKN1C are implicated in sporadic can- types of the human placenta [125, 126], which also correlates cers and Beckwith-Wiedemann syndrome suggesting that with placental HLX expression from our findings [74]. This it is a tumor suppressor candidate. Larson et al. [117] current study showed that CCNB1 mRNA expression was have suggested that decreased CDKN1C expression may be significantly reduced in cultured trophoblast cells with HLX involved in human breast carcinogenesis in vivo. CDKN1C inactivation, suggesting that CCNB1 to be a downstream has recently been recognized as a maternally imprinted gene, target gene of HLX. CCNB1 expression was also significantly supporting its role for genomic imprinting in the regulation reduced in FGR-affected placentae compared with controls. of embryonic implantation and development and placental Therefore, our findings showed that the cell culture model growth, as well as in the pathogenesis of proliferative was consistent with the observed changes seen in HLX trophoblastic diseases [118]. In the normal placenta, strong levels in FGR and changes in CCNB1 levels and suggested nuclear CDKN1C expressionwasobservedinextravillous a causative role between reduced HLX levels and reduced trophoblast, cytotrophoblast, and implantation-site inter- CCNB1 levels in FGR. stitial trophoblast, but was absent in syncytiotrophoblast JUN is a member of the AP-1 family of transcription [118]. This expression of CDKN1C in the human placenta factors and is implicated as a key regulator of human is consistent with the expression pattern of HLX from our extravillous trophoblast proliferation, invasion, and differen- study [74]. tiation [127]. Not surprisingly, JUN is also strongly expressed Studies have shown that targeted disruption of CDKN1C in the highly proliferative extravillous trophoblast cells of in the mouse model system results in severe placental the human placenta [127], consistent with HLX expression defects [119]. CDKN1C knock-out mice have displayed in the human placenta [74]. JUN plays a key role in an array of pre-eclampsia symptoms, including placental coordinating steroid hormone actions in a variety of tissues abnormalities, hypertension, proteinuria, and premature [128] and is induced by the steroid hormone oestrogen in labour [119]. Results from our own findings showed that the human endometrium. Salmi and Rutanen [129]have CDKN1C expression is significantly reduced in cultured demonstrated a strong expression of JUN in human prolif- trophoblast cells, therefore, is a direct or indirect target candidate gene of HLX in cultured trophoblast cells [108]. erative endometrium and that JUN expression is decreased This suggests that HLX-mediated reduction of CDKN1C in the human decidua throughout pregnancy [130]. expression may reduce trophoblast proliferation. Further Our own findings showed significantly decreased JUN confirmation of CDKN1C mRNA expression in FGR- expression with HLX gene reduction in cultured trophoblast affected human placentae, also show a significant decrease cells. This suggests, as a downstream target of HLX, JUN is in human idiopathic FGR compared with gestation-matched regulated by HLX, either directly or indirectly, in order to controls. affect proliferation, invasion, and differentiation of extrav- ELK1, as a member of the ETS family, acts as a illous trophoblasts. Consistently, results showed that JUN transcriptional factor for the MET gene [120], which is expression was also significantly decreased in FGR-affected expressed in placental cytotrophoblasts [121]. As with HLX, human placentae compared with control placentae. There- ELK1 is also expressed in human extravillous trophoblast fore, decreased JUN expression, either directly or indirectly cells [122] and is suggested to play a role in the regulation by HLX, may result in anomalous trophoblast functions asso- of cell proliferation and migration [123]. ETS transcription ciated with FGR. Furthermore, HLX-mediated JUN dysreg- factors are also critical for human uterine decidualisation ulation of trophoblast differentiation and invasion can lead [124]. Human decidual fibroblasts expressed significantly to abnormal spiral artery remodeling by endovascular tro- less mRNA for the decidualisation markers prolactin, IGFBP- phoblasts, as these endovascular trophoblasts need to differ- I, EBAF, TIMP3, decorin, and laminin in the presence of entiate from cytotrophoblasts and invade the maternal spiral an antisense oligonucleotide that blocks the translation of arteries for enhanced blood flow during pregnancy [16]. ETS mRNA when compared with decidual fibroblast cells Thus, the candidate downstream target genes of a exposed to a control oligonucleotide [124]. homeobox gene, HLX, are significantly altered in human Given our previous findings of HLX expression in the idiopathic FGR-affected placentae, compared with gestation- human placenta [74] and in the importance of HLX in matched controls. Most importantly, the findings of our own trophoblast proliferation [77] and migration [76], ELK1 study demonstrated that in vitro models for siRNA-mediated is a potential target gene of HLX in the control of knockdown of HLX expression in placental trophoblast cells trophoblast cell proliferation and migration. Consistently, show consistent changes to those observed in human FGR results from our observation showed that ELK1 expression where HLX levels are reduced. These results suggest that was also significantly decreased in FGR-affected human reduced levels of HLX seen in FGR cause direct or indirect placentae. Therefore, the cell culture model where siRNA- effects on target genes that have been shown to be altered mediated reduction of HLX reduces ELK1 was consistent in FGR. Therefore, reduced HLX levels directly or indirectly with decreased levels of HLX and decreased levels of ELK1 cause gene expression changes in targets that have deleterious in human FGR. effects on trophoblast function. Journal of Pregnancy 7

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Minerva Access is the Institutional Repository of The University of Melbourne

Author/s: Murthi, P; Rajaraman, G; Brennecke, SP; Kalionis, B

Title: The role of placental homeobox genes in human fetal growth restriction.

Date: 2011

Citation: Murthi, P., Rajaraman, G., Brennecke, S. P. & Kalionis, B. (2011). The role of placental homeobox genes in human fetal growth restriction.. J Pregnancy, 2011, pp.548171-. https://doi.org/10.1155/2011/548171.

Persistent Link: http://hdl.handle.net/11343/263870

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